Four years after the disaster at Jurassic Park, the wealthy Bowman family makes an excursion to the island of Isla Sorna during a yachting cruise. Their young daughter wanders off and is attacked by a pack of Compsognathus, but survives. The incident allows Peter Ludlow, John Hammond's nephew, to gain control of his uncle's company InGen, which is in severe financial straits after the events at Jurassic Park. Hammond contacts Ian Malcolm and explains that Isla Sorna is the island where the dinosaurs were engineered and nurtured before being moved to Isla Nublar, Jurassic Park's location. He also explains that after the park was shut down, Isla Sorna was abandoned during a hurricane and the dinosaurs left to survive in the wild, where they have been inexplicably thriving. Hammond asks Malcolm to join a team that will travel to Isla Sorna to document the dinosaurs in order to rally public support against human interference on the island. Ian initially declines but, after learning that his girlfriend, paleontologist Sarah Harding, is part of the team and is already on the island, he agrees to go.
Ian meets his teammates Eddie Carr, an equipment specialist and engineer, and Nick Van Owen, a video documentarian. After arriving on the island, they find Sarah and discover that Ian's daughter Kelly has stowed away on their trailer. They then watch as an InGen team of mercenaries, hunters and paleontologists led by Ludlow arrive to chase and capture several dinosaurs. Meanwhile, tracker and team leader Roland Tembo separates from the main group and tries to go after a male Tyrannosaurus rex. He decides to capture it by luring it to the cries of its injured offspring. That night, Ian's team sneak into the InGen camp and learn the captured dinosaurs will be brought to San Diego as the main attraction of a newly proposed theme park. This prompts Nick and Sarah to free the caged dinosaurs, wreaking havoc upon the camp, leaving the InGen team realizing they are not alone on the island.
During the commotion, Nick frees the infant T. rex and takes it to the trailer to mend its broken leg. After securing Kelly in a shelter with Eddie, a loud roar from a T. rex is heard in the distance and Ian realizes the infant's parents are searching for it and rushes to the trailer. As soon as he arrives, the infant's parents, two adult T. rexes emerge on both sides of the trailer, and Ian, Sarah, and Nick release the infant. However, the two adult T. rex then attack the trailer, pushing it over the edge of a nearby cliff. Eddie soon arrives and manages to save the others, but as he tries to pull the trailer back over the edge with his SUV, the two adult T. rexes return to the trailer's location and he is devoured by the duo, destroying the team's equipment. Ian, Sarah, and Nick are found and pulled off the cliff by the InGen team, along with Kelly. With both groups' communications equipment and vehicles destroyed, they team up to reach the old InGen compound's radio station on foot.
The next night, the two adult T. rexes come across the group's camp by tracking the scent of the T. rex infant's blood that was on Sarah's shirt. A frightened group member notices the "T. rex" and causes everyone to flee in panic. The female T. rex chases the group to a waterfall cave, while Roland stays behind and manages to tranquilize the male. The remains of the team flee through a tall grass savannah, where a pack of Velociraptors ambushes them. When Ian, Sarah, Nick, and Kelly arrive, Ian realizes that the Velociraptors are nearby, and they hurry toward the compound. Nick runs ahead to the InGen Worker's Village, where he discovers the communications center and calls for rescue. When Ian, Sarah and Kelly arrive they are attacked by a trio of Velociraptors. The three of them manage to evade the Raptors until a helicopter finally arrives and transports them off the island. From the air, they spot the unconscious male T. rex being prepared for transport.
A freighter carries the T. rex back to the mainland, but crashes into the dock. An investigation finds the entire crew dead. A guard opens the cargo hold, accidentally releasing the conscious T. rex, which escapes into the city of San Diego and goes on a rampage. Realizing the T. rex is likely searching for its infant, Ian and Sarah learn from Ludlow that the infant was captured and is in a secure InGen building. They retrieve the infant and use it to lure the adult back to the ship. Ludlow tries to intervene but is trapped and cornered in the cargo hold by the adult T. rex and mauled by the infant. Before the adult can escape again, Sarah tranquilizes it while Ian closes the hold.
Ian, Sarah, and Kelly watch on live television as the ship carrying the adult and infant T. rex is escorted back to Isla Sorna. Hammond explains in an interview that the American and Costa Rican governments have agreed to declare the island a nature preserve. He ends the interview by saying "life will find a way". The ending shows the two adult T. rexes at peace with the infant along with a herd of Stegosaurus and a flock of Pteranodons nearby.
- Stephen, Zecora, Steamy, Puffy, Evan, Shai-Shay, Sharky, Willy (the train), the Miner Trains (Steam Grindor, Steam Mech, Mucker, Steam Claw D., Steam Driller, Steam Sweeper, and Steam Exavator), T.C., Hugs, Buzz, J.J., Mako, and Rattlesnake Jake will guest star in this film.
- The storyline continues in Thomas and Twilight Sparkle visit Jurassic Park III.